PHOSPHORYLATION OF SER(232) DIRECTLY REGULATES THE TRANSCRIPTIONAL ACTIVITY OF THE P-PROTEIN OF HUMAN RESPIRATORY SYNCYTIAL VIRUS - PHOSPHORYLATION OF SER(237) MAY PLAY AN ACCESSORY ROLE

The phosphoprotein P of human respiratory syncytial virus (RSV) was ex pressed in eukaryotic cells in phosphorylated form. Site-directed muta genesis of the recombinant protein established Ser(232) as the major s ite of phosphorylation in vivo. Phosphorylation of bacterially made P protein in vitro by purified casein kinase II (CKII) resulted in the p hosphorylation of Ser(237), whereas mainly Ser(232) was phosphorylated by a crude cell extract. The P kinase activity in the cell extract ex hibited properties characteristic of CKII. While the Ser(232,237) to A la double mutant was nearly completely defective for phosphorylation a nd transcription, phosphorylation at Ser(232), through the use of appr opriate P mutant or kinase, activated P protein. Phosphorylation of Se r(237) restored activity only to the extent it facilitated phosphoryla tion of Ser(232). Phosphate groups of P protein in RSV-infected cells were highly stable; inhibitors of protein serine phosphatases had no e ffect on the intracellular turnover of the phosphates. Highly purified viral polymerase L was transcriptionally active but devoid of P prote in kinase activity. Thus, CKII-mediated phosphorylation of Ser(232) ap pears to be the primary regulator of P protein activity while phosphor ylation of Ser(237) may be involved in a modulatory role under certain conditions. (C) 1995 academic Press, Inc.